In verde le connessioni fra alcune cellule del centro della respirazione nel tronco encefalico e aree superiori del cervello (Cortesia Kevin Yackle et al./Science/AAAS)

I neuroni che collegano respirazione e calma – Breathing control center neurons that promote arousal in mice. The calming effect of breathing. Breathing to inspire and arouse.

Un minuscolo gruppo di neuroni situato nella parte del tronco encefalico che controlla la respirazione comunica direttamente con una struttura cerebrale coinvolta nelle risposte allo stress. La scoperta potrebbe indicare le basi fisiologiche degli effetti calmanti della meditazione e avere ricadute sullo sviluppo di farmaci contro gli attacchi di panico e altri disturbi

E’ un piccolo gruppo di neuroni nel tronco cerebrale a regolare i rapporti fra la respirazione e le attività cerebrali superiori connesse a uno stato di calma oppure di agitazione.
La scoperta è di un gruppo di ricercatori della Stanford University, che firmano un articolo pubblicato su “Science” http://science.sciencemag.org/content/355/6332/1411 . In prospettiva, il risultato può avere ricadute in campo clinico con lo sviluppo di nuove terapie contro gli attacchi di panico e altri disturbi legati a stati di stress.

Anche se la respirazione è generalmente considerata un comportamento controllato soprattutto dal sistema nervoso autonomo, l’esistenza di strette connessioni con le aree cerebrali che presiedono alle funzioni cerebrali superiori è ben nota e ampiamente testimoniata, per esempio, dagli studi sugli effetti della meditazione, che ha uno dei suoi cardini proprio nel controllo della respirazione.
Tuttavia finora non era chiaro quali fossero i centri e i meccanismi neuronali che presiedono ai rapporti fra respiro e cervello.
ImageSource / AGFIn uno studio sperimentale sui topi, Kevin Yackle e colleghi hanno ora identificato il regista di questi rapporti in un piccolo gruppo di neuroni situato nel tronco cerebrale.
Si tratta in particolare di circa 175 neuroni del cosiddetto complesso di pre-Bötzinger, un articolato gruppo di 3000 neuroni circa la cui attività ritmica avvia i movimenti respiratori. I neuroni di questa sottopopolazione inviano delle proiezioni direttamente a un’area del cervello, il locus coeruleus, che ha un  ruolo chiave nello stato di vigilanza in generale, nella focalizzazione dell’attenzione, e nelle risposte allo stress.
Dopo aver eliminato in alcuni topi i neuroni identificati, i ricercatori hanno constatato che la loro respirazione era rimasta perfettamente normale, ma che gli animali rimanevano insolitamente tranquilli anche se erano sottoposti a stimoli che normalmente inducono una risposta di stress.

Dato che questi neuroni possono essere identificati grazie alla presenza di specifici marcatori molecolari, i ricercatori sperano che sia possibile sviluppare in tempi relativamente brevi farmaci in grado di agire selettivamente su di essi. http://www.lescienze.it/news/2017/03/31/news/neuroni_respirazione_stress_cervello-3477488/

Breathing control center neurons that promote arousal in mice

Kevin Yackle1,*, Lindsay A. Schwarz2,†, Kaiwen Kam3,4, Jordan M. Sorokin5, John R. Huguenard5, Jack L. Feldman3, Liqun Luo2, Mark A. Krasnow1,‡

1. 1Howard Hughes Medical Institute, Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
2. 2Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, CA 94305, USA.
3. 3Systems Neurobiology Laboratory, Department of Neurobiology, David Geffen School of Medicine, University of California–Los Angeles, Los Angeles, CA 90095, USA.
4. 4Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
5. 5Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305, USA.

1. ↵‡Corresponding author. Email: krasnow@stanford.edu

• ↵* Present address: Department of Physiology, University of California–San Francisco, San Francisco, CA 94158, USA.

• ↵† Present address: Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA.

Science  31 Mar 2017: Vol. 355, Issue 6332, pp. 1411-1415. DOI: 10.1126/science.aai7984

The calming effect of breathing

The rhythmic activity of a cluster of neurons in the brainstem initiates breathing. This cluster is composed of distinct, though intermingled, subgroups of neurons. Yackle et al.found a small, molecularly defined neuronal subpopulation in this breathing rhythm generator that directly projects to a brain center that plays a key role in generalized alertness, attention, and stress (see the Perspective by Sheikbahaei and Smith). Removal of these cells did not affect normal breathing but left the animals unusually calm. The breathing center thus has a direct and dramatic influence on higher-order brain function.
Abstract

Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

Although breathing is commonly viewed as a simple autonomous function that sustains life, it has long been known to influence higher-order behavior and thinking (1). Slow, controlled breathing is used by practitioners of pranayama yoga and other forms of meditation to promote mental calming and contemplative states, and it is used clinically to suppress excessive arousal and stress such as certain types of panic attacks (2, 3). Although the effect of breathing on behavior and mental state could easily be indirect, there could also be more direct connections and impact of the breathing center on higher-order brain function (4), as demonstrated here.

The preBötzinger complex (preBötC) is a cluster of several thousand neurons in the ventrolateral medulla of the murine brain that can autonomously generate respiratory rhythms in explanted brain slices (5, 6), and whose rhythmic activity in vivo initiates breathing by recurrently activating premotor and motor neurons of the respiratory muscles (5). The preBötC is not a homogeneous population of neurons but is composed of distinct, though intermingled, neuronal subpopulations (5, 7), one of which is essential for respiratory-rhythm generation (8, 9) and another for sighing (10).

To systematically explore the molecular diversity of breathing center neurons, we screened expression patterns of over 19,000 genes in the Euroexpress embryonic day 14.5 (E14.5) mouse hindbrain database (11). Cadherin-9 (Cdh9) was the gene most selectively expressed in preBötC (Fig. 1A). We constructed a bacterial artificial chromosome (BAC) transgene with the mOrange coding sequence inserted at the Cdh9 translation start codon (Fig. 1B). Cell counts in early postnatal brains detected 319 ± 130 (n = 6) Cdh9-mOrange–expressing cells in preBötC (Fig. 1C). These cells intermingled with neurons expressing canonical preBötC markers somatostatin (SST) and neurokinin 1 receptor (NK1R) (Fig. 1, D and E, and fig. S1, A to E). Few expressed the markers themselves: 0 out of 43 Cdh9-mOrange+ cells scored were SST+, and 7 out of 179 Cdh9-mOrange+ (4%) were NK1R+. All Cdh9-mOrange–expressing cells coexpressed neuronal marker NEUN (n = 61 cells, Fig. 1F). These Cdh9-mOrange–expressing neurons can be further divided into seven subtypes based on differential expression of transcription factors Pax2, Dach1, Lmo4, Evx1, and Dbx1. We focused on the ~175 neurons in each preBötC (fig. S1, F to J; ~350 neurons bilaterally) that coexpress the Dbx1-lineage marker, the major subpopulation (56%, 165 out of 292 scored Cdh9-mOrange+ cells, were Dbx1-LacZ+) we call Cdh9/Dbx1 neurons (Fig. 1, G to I).

We electrophysiologically recorded 26 mOrange-positive neurons in 15 preBötC slice preparations from Cdh9-mOrange;Dbx1-lacZ double-transgenic postnatal day 0 to 5 (P0 to P5) mice, then poststained the neurons for β-galactosidase (LacZ) in some preparations to identify recordings of Cdh9/Dbx1 neurons (table S1). We definitively identified five Cdh9/Dbx1 neurons. One showed bursts of action potentials just before or during each preBötC inspiratory burst (Fig. 1J and fig. S2), like most other Dbx1-lineage preBötC neurons (12). Three other neurons were more broadly active with bursts during some, but not all, preBötC inspiratory bursts (42, 41, and 88% of inspiratory bursts; Fig. 1K and fig. S2), called an “inspiratory-associated” activity pattern. The other neuron showed sporadic activity with no apparent relationship to preBötC inspiratory bursts. Seven of the 15 Cdh9-mOrange neurons whose Dbx1-lacZ expression status was not determined also displayed inspiratory (four neurons) or inspiratory-associated (two neurons) patterns (table S1).

We used intersectional genetics (Cdh9-LOSL-DTR;Dbx1-cre) (Fig. 1, B, M, and N) to express human diphtheria toxin receptor (DTR) only in Dbx1-lineage cells that coexpress Cdh9, so that Cdh9/Dbx1neurons could be specifically ablated by intraperitoneal injection of diphtheria toxin. We expected there would be few, if any, cells besides Cdh9/Dbx1 preBötC neurons that express both genes (11, 13). We examined this in two ways. First, we compared mOrange expression in Cdh9-LOSL-DTR and Cdh9-LOSL-DTR;Dbx1-cre transgenic mice by immunostaining serial sections of adult brains. The only regions where Dbx1-cre reduced the number of mOrange-expressing cells were preBötC and inferior colliculus (fig. S3). Costaining with other markers showed that loss of mOrange in preBötC was specific and complete for Cdh9/Dbx1 neurons and had little or no effect on the six other (Dbx1-negative) Cdh9-positive preBötC cell types (fig. S4). Second, immunostaining of serial sections of Cdh9-LOSL-DTR;Dbx1-cre adult brains detected DTR expression only in Cdh9/Dbx1preBötC neurons (Fig. 1M) and the inferior colliculus (fig. S3). Intraperitoneal injection of diphtheria toxin once a day for 3 days eliminated the DTR-expressing cells (Fig. 1N).

We analyzed adult mice several days after ablating Cdh9/Dbx1 neurons. We expected Cdh9/Dbx1neurons would be essential for breathing and viability because Dbx1 neurons are essential for breathing in vivo (8, 9) and ablating just 85 random Dbx1 neurons abolishes preBötC rhythms in vitro (14). However, there was no overt effect on viability (three of three scored mice alive >1 year after ablation), breathing, or sensory and motor behaviors. Plethysmography of freely moving adult mice after ablation did not detect significant differences in inspiratory time, expiratory time, or tidal volume of standard (eupneic) breaths (figs. S5A and S6). No differences were detected in characteristics of four variant breath types (figs. S5, B to E, and S6) or breaths during sleep or under hypercapnic or hypoxic conditions (figs. S5, F to H, and S6).

There was, however, a change in abundance of different breath types, first noted in respiratory-rate histograms: Cdh9/Dbx1 ablation shifted the distribution toward slower breaths (low respiratory rate) (Fig. 2B). Inspection of plethysmograph traces indicated that the shift was due to increased slow breaths (eupneic and grooming) associated with calm behaviors and reduction in rapid breaths associated with sniffing and other active behaviors (Fig. 2A and fig. S7).

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The change in breathing patterns after Cdh9/Dbx1 neural ablation was accompanied by a corresponding change in behavior. Ablation reduced exploration of a new environment (87 ± 9% versus 62 ± 20% time spent in active exploration, pre- versus postablation; P = 0.02) and increased time engaged in grooming threefold (10 ± 7% versus 31 ± 16%; P = 0.02) and still-sitting twofold (3 ± 4% versus 7 ± 7%; P = 0.07), although the latter did not reach statistical significance (Fig. 2C). There was an increase in both number of calm episodes (Fig. 2D) and their duration (Fig. 2E and movie S1). Because the breathing pattern associated with each behavioral state was not detectably altered by ablation (figs. S5 and S6), the observed change in respiratory-rate distribution could be explained by the overall change in behavior (fig. S8): a shift from active toward calm behaviors. Electrocorticographic (ECoG) monitoring showed an increase in slow-wave (delta, 2 to 4 Hz) brain activity after ablation (Fig. 2F) and a selective decline in time spent in an active-brain state dominated by theta activity (Fig. 2G) compared to that of littermate controls (Fig. 2, H and I). These changes could be temporarily reversed by illuminating the chamber, providing a stimulus that apparently overrides the decrease in arousal caused by Cdh9/Dbx1 ablation (fig. S9).

To confirm that the observed behavioral and breathing changes were due to ablation of neurons in the preBötC, we restricted DTR induction and hence neural ablation to just Cdh9-expressing preBötC neurons (fig. S10, A to C). The animals displayed a diminution in active exploratory behaviors and breathing patterns and an increase in calm behaviors and breathing patterns similar to animals with Cdh9/Dbx1 neurons ablated using our intersectional genetic strategy (compare fig. S10, D and E, and Fig. 2).

The decrease in active behavior and increase in ECoG delta waves observed after Cdh9/Dbx1neuron ablation is reminiscent of changes following silencing or ablation of the locus coeruleus (LC), a noradrenergic nucleus in the pons implicated in generalized arousal, stress, and sleep-wake transitions (15). We thus microinjected two retrograde tracers into the LC and found that both labeled Cdh9-mOrange neurons in preBötC (FluorGold: fig. S11, A to C; fluorescent retrograde beads: data not shown). Most of the labeled Cdh9-mOrange preBötC neurons (85%, 23 out of 27, n= 40 sections, 3 mice) were contralateral to the injection site. The connection is selective because most labeled preBötC neurons expressed Cdh9-mOrange (72%, 13 out of 18, n = 10 sections, 4 mice), and no retrograde labeling of Cdh9-mOrange neurons was observed following tracer injection into regions surrounding the LC (n = 4 injections).

We tested whether the observed connection between the preBötC neurons and LC is direct to the noradrenergic [dopamine β-hydroxylase (Dbh)–expressing] neurons that dominate the LC (15). Injection of Cre-dependent adeno-associated virus (AAV) helper viruses (AAV-FLEx^loxP-TVA:mCherry and AAV-FLEx^loxP-rabies glycoprotein), which enables infection and monosynaptic spread of an envA-pseudotyped, glycoprotein-deleted, and GFP-expressing rabies virus (RVdG) (16), into the LC of Dbh-cre;Cdh9-mOrange mice resulted in specific infection of LC “starter” neurons by RVdG (fig. S12) and selective retrograde transsynaptic tracing (GFP-labeling) of Cdh9-mOrange neurons in the preBötC (Fig. 3, A to D, and fig. S11, D to F). We again observed a strong bias for labeling the contralateral rather than ipsilateral preBötC (compare Fig. 3, A to C, and fig. S11, D to F). Most of the green fluorescent protein (GFP)–labeled neurons in the contralateral preBötC (63%, 45 out of 72, n = 18 sections, 3 mice) expressed Cdh9-mOrange, whereas few of the labeled ipsilateral preBötC neurons expressed Cdh9-mOrange (7%, 7 out of 94, n = 18 sections, 3 mice). Nearly all ipsilateral GFP-expressing neurons were located outside the canonical preBötC region defined by SST expression, so they are presumably not preBötC neurons (fig. S13). Control experiments in mice without Dbh-cre did not show double-positive preBötC neurons (fig. S12).

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We microinjected a retrogradely transported cre-expressing virus, canine adenovirus type 2–Cre (CAV-Cre), bilaterally into the LC of Cdh9-LOSL-DTR mice to eliminate mOrange and express DTR only in the Cdh9-expressing neurons that project to LC (Fig. 3E). Before DT injection, animals breathed and behaved normally, although there was an increase in calm breathing relative to mock-ablated controls, perhaps because of neural toxicity associated with induced DTR expression (Fig. 3, H and I). Four to 10 days after DT injection and ablation of LC-projecting, Cdh9-expressing preBötC neurons (Fig. 3, F and G), there was a change in breathing (Fig. 3H) and decrease in active behaviors (Fig. 3I) that mimicked those observed after ablation of the Cdh9/Dbx1 preBötC neurons (compare to Fig. 2, B and C).

We examined LC activity by c-FOS expression (17) following Cdh9/Dbx1 neuron ablation. Under standard housing conditions, Cdh9-LOSL-DTR;Dbx1-cre littermate control mice with intact Cdh9/Dbx1 neurons showed only occasional LC activity (Fig. 4, A and D, and fig. S14). When animals were placed in a new environment (plethysmography chamber) for 1 hour, c-FOS was induced in scattered cells throughout the LC (Fig. 4, B and D, and fig. S14). However, 4 days after Cdh9/Dbx1 neuron ablation, only rare c-FOS–positive LC neurons were detected before or after placement in the chamber (Fig. 4, C and D, and fig. S14). The LC of ablated animals remained responsive to the extreme arousal stimulus of physical-restraint stress (18) (Fig. 4, D to F, and fig. S13), indicating that other LC inputs and functions were intact.

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We have identified and characterized a new neuronal subtype in the preBötC comprising ~175 of its ~3000 neurons. The Cdh9/Dbx1 neurons are dispensable for respiratory-rhythm generation and instead promote generalized behavioral arousal. Ablation of these neurons left all major breathing patterns and regulation intact but made the mice preternaturally calm: Their activity and ECoG profiles shifted from active exploratory behavior and brain wave patterns toward calm behaviors such as still-sitting and grooming. Monosynaptic tracing demonstrated that these neurons directly project to and synapse on noradrenergic neurons in the contralateral LC, which in turn project throughout the brain and control generalized arousal and sleep-wake transitions (15). Cdh9/Dbx1preBötC neurons provide excitatory input to the LC and appear to be the dominant activating input under mild arousal conditions of placement in a new chamber.

We propose that Cdh9/Dbx1 preBötC neurons function as gateway neurons directly linking the preBötC to the locus coeruleus, and through it to the rest of the brain (Fig. 4G). This ascending circuit allows the respiratory center to communicate directly with and control higher-order brain structures associated with behavioral arousal. The excitatory input to the LC is presumably provided by the observed inspiratory-associated activity patterns of Cdh9/Dbx1 neurons, which could provide greater excitatory input with faster respiratory rates and perhaps abnormal respiratory patterns (Fig. 4H). This respiratory corollary signal would thus serve to coordinate the animal’s state of arousal with the breathing pattern, leaving the animal calm and relaxed when breathing is slow and regular, but promoting (or maintaining) arousal when breathing is rapid or disturbed. This circuit and corollary signal would explain why preBötC respiratory patterns have been observed in the LC and other reticular activating structures (19–21). The LC can increase respiratory rate (22), so there may also be a positive feedback loop from the LC ultimately back to Cdh9/Dbx1 preBötC neurons.

The Cdh9/Dbx1 circuit may have evolved as a defense response, mobilizing the animal in the face of rapid, irregular, or labored breathing. Indeed, fast or erratic breathing in humans increases alertness and can cause anxiety and even panic (3), and likewise increased preBötC activity, hyperventilation, and sighs appear to induce arousal during sleep (23–25). Conversely, slow and controlled breathing has long been known by practitioners of pranayama yoga to induce relaxation, and related approaches have proven useful for anxiety syndromes and other stress disorders (1, 2). If the Cdh9/Dbx1 circuit is conserved in humans, it could provide a therapeutic target for breathing-related anxiety disorders and perhaps prevention of sudden infant death syndrome (SIDS), widely hypothesized to result from an inadequate arousal response to asphyxiation during sleep (24). Notably, panic attacks triggered by respiratory symptoms are specifically responsive to clonidine, an α2-adrenergic agonist that silences LC (26).

Although breathing is generally thought of as an autonomic behavior, higher-order brain functions can exert exquisite control over breathing. Our results show, conversely, that the breathing center has a direct and powerful influence on higher-order brain function. It will thus be important to map the full range of behaviors and functions the breathing center controls. https://science.sciencemag.org/content/355/6332/1411

Breathing to inspire and arouse

Science  31 Mar 2017:
Vol. 355, Issue 6332, pp. 1370-1371
DOI: 10.1126/science.aan1466

Summary

Breathing is one of the perpetual rhythms of life that is often taken for granted, its apparent simplicity belying the complex neural machinery involved. This behavior is more complicated than just producing inspiration, as breathing is integrated with many other motor functions such as vocalization, orofacial motor behaviors, emotional expression (laughing and crying), and locomotion (1, 2). In addition, cognition can strongly influence breathing. Conscious breathing during yoga, meditation, or psychotherapy can modulate emotion, arousal state, or stress (3). Therefore, understanding the links between breathing behavior, brain arousal state, and higher-order brain activity is of great interest. On page 1411 of this issue, Yackle et al. (4) identify an apparently specialized, molecularly identifiable, small subset of ∼350 neurons in the mouse brain that forms a circuit for transmitting information about respiratory activity to other central nervous system neurons, specifically with a group of noradrenergic neurons in the locus coeruleus (LC) in the brainstem, that influences arousal state (see the figure). This finding provides new insight into how the motor act of breathing can influence higher-order brain functions. https://science.sciencemag.org/content/355/6332/1370